Modern agricultural practice relies heavily on the use of insecticides and nematicides to improve crop yields. The vast majority of pesticides are chemical agents. As has been widely recognized, the use of chemical pesticides has a number of disadvantages. Conventional chemical insecticides frequently act nonspecifically, killing beneficial insect species in addition to the intended target. Chemicals may persist in the environment and present a danger to organisms higher up in the food chain than the insect pest. Exposure to chemical pesticides is hazardous and poses a threat to the health of agricultural workers and others. In addition, with repeated applications of pesticides resistant pest populations frequently emerge.
Biological pest control agents provide an attractive alternative to the use of chemicals. The term "biological pest control agent" refers to a naturally occurring agent that, when contacted with a pest, is able to infect the pest and interfere with its normal functioning so as to bring about the incapacitation or death of the pest. As used herein, "biological pest control agents" include naturally occurring agents that have been modified in certain ways. In the context of insect control, such agents comprise primarily viruses and bacteria. Insect control strategies utilizing biological control agents take advantage of the naturally occurring interactions between the agent and its insect hosts. The use of viruses and bacteria as pest control agents capitalizes on features such as the ability to enter target cells and exert pathogenic effects.
Biological pest control agents have the potential to be much more specific in terms of their targets than chemical pesticides affecting only insects within the host range of the virus or bacterium. Specificity, while desirable from the standpoint of maximizing safety and minimizing environmental impact, is also a potential disadvantage. In most settings multiple pest species are present, and it is not cost effective to deliver multiple different insecticides rather than a single agent with a broad spectrum of activity. The present invention addresses this and other limitations of prior art biological pest control agents.
A wide variety of viruses selectively infect insects, and the use of many of these as potential insect control agents has been explored (discussed in Miller, L. and Ball, L. A., eds., The Insect Viruses, New York, 1998). In particular, use of baculoviruses as biological insect control agents has been the subject of much research over the past three decades. Baculoviruses are double-stranded DNA viruses that specifically infect arthropods. Most baculoviruses under consideration as biological insect control agents have host ranges limited to the order Lepidoptera. In a typical baculovirus infection, larvae feeding on a plant contaminated with virus ingest the virus. If a susceptible insect ingests sufficient quantity of virus, a productive systemic infection ensues. Infection is most severe if established during an early larval stage. Depending on the stage of infection and other variables such as dose, temperature, and nutrition, death usually occurs within one to ten days, most commonly between four and seven days.
The baculovirus replication cycle has been extensively studied (reviewed in Miller, L. ed., The Baculoviruses, New York, 1997, the contents of which are incorporated herein by reference). The first step is adsorption of the virus to the surface of a host cell. A specific molecular target on the cell surface has not been identified. After ingestion by an insect, entry through the midgut is believed to occur by direct fusion of the viral envelope with the cell membrane. Uncoating of the viral DNA occurs following viral entry. In host cells able to support a productive infection, the viral DNA enters the cell nucleus, where gene expression takes place.
Baculovirus genes are expressed sequentially, in a temporally regulated manner. Baculovirus genes are divided into immediate-early, delayed early, late, and very late genes based upon the phase of the viral replicative cycle in which they are expressed. Temporal control is likely achieved by a cascade of transcriptional regulation in which the transcription of later genes depends upon the presence of gene products produced earlier. Transcription of immediate-early genes begins soon after viral entry into the nucleus and does not require viral gene products, relying instead on host transcriptional machinery. Temporal control of transcription is a characteristic of many viruses.
One well-characterized baculovirus immediate-early gene is known as IE1. Other immediate early gene promoters include IEN and IE0. Examples of very late genes include the polyhedrin and P10 genes. Expression from the polyhedrin and P10 promoters requires the activity of viral gene products produced at earlier stages of the replicative cycle. Thus genes under the control of the polyhedrin or P10 promoters can occur only in baculovirus-infected cells during the very late phase of the infection (Guarino et al., U.S. Pat. Nos. 5,077,214 and 5,162,222 and references therein).
In addition to promoter elements the baculovirns genome contains enhancer elements that enhance transcription from promoters to which they are linked. In particular, the enhancer elements hr1, hr2, hr3, hr4, and hr5 enhance expression from immediate early gene promoters (Guarino et al., U.S. Pat. Nos. 5,077,214 and 5,162,222 and references therein).
Baculoviruses occur in two forms. Mature viral particles (nucleocapsids) can be packaged in occlusion bodies within the nucleus of infected cells. Occlusion bodies consist primarily of polyhedrin, a viral protein expressed very late in the replicative cycle, and nucleocapsids. Occlusion bodies, typically containing large numbers of viral particles are released into the environment upon death of the host cell or insect and are able to withstand environmental stresses for long periods of time. The occlusion body structure thus protects the virus until ingestion by a susceptible host and mediates transmission from one organism to another. Baculoviruses can also bud from the membrane of the host cell, acquiring a membranous envelope as they do so. This so-called nonoccluded form is the means by which the virus spreads from cell to cell within the host. Nonoccluded viral particles are rapidly degraded upon exposure to the environment. In the absence of polyhedrin, e.g., in the case of infection by baculoviruses lacking the polyhedrin gene, only nonoccluded viral particles are produced.
Recombinant baculoviruses are widely used for the production of foreign proteins in insect cell lines. Rather than modifying the viral genome directly, creation of recombinant baculoviruses utilizes transfer plasmids. These plasmids generally contain sequences that permit them to replicate in bacteria, allowing convenient manipulation. In addition, they contain a baculovirus promoter capable of driving expression of a foreign gene operably linked to the promoter and a multiple cloning site into which such a foreign gene can be inserted. Some transfer plasmids incorporate additional genetic control elements such as enhancers and transcriptional terminators. Selectable markers such as drug resistance genes and markers whose presence can be readily detected, e.g., the beta-galactosidase gene, may be included to facilitate molecular biological manipulations and selection of recombinant baculoviruses. The multiple cloning site and genetic control elements are flanked by sequences homologous to a portion of the baculovirus genome, thus targeting the plasmid for insertion into baculovirus DNA. Transfer plasmid can be used to produce either occlusion-positive or occlusion-negative baculovirus recombinants depending upon whether a functional polyhedrin gene is present in the recombinant.
There is evidence that baculovirus diseases occur in at least 500 different insect species including many of the most significant pests of agricultural and forestry crops. However, individual baculovirus strains are usually restricted in their replication to one or a few insect species. Thus pathogenic effects that require viral replication can be targeted to the species for which control is sought. Some baculoviruses, e.g., members of the Autographa califomica nuclear polyhedrosis virus (AcNPV) family, can replicate at some level in a wide variety of Lepidopteran hosts.
Although highly attractive from the standpoint of environmental safety, development of baculoviruses as biological insect control agents has proven problematic. The generally prolonged time from viral exposure to death, during which larvae continue to damage the plant, has meant that in most cases unmodified baculoviruses are not viable alternatives to chemicals, which halt larval crop damage almost immediately upon contact.
Three general approaches have been employed in attempts to improve the efficacy of baculoviruses as pest control agents by genetically modifying the virus. One approach has been to utilize viruses in which certain baculovirus genes that may prolong the life of the host have been inactivated or deleted from the viral genome (e.g., U.S. Pat. No. 5,858,353). A second approach has been to use the virus to direct inappropriate expression of insect genes that normally regulate important aspects of insect physiology or development, with the goal of interfering with key physiological or developmental processes such as fluid balance or molting. Among the genes that have been utilized are those encoding diuretic hormone, juvenile hormone esterase, and prothoracicotropic hormone (reviewed in Black et al., "Commercialization of Baculoviral Insecticides" in Miller, L. (ed.), The Baculoviruses, 1997). A third approach has been to engineer baculoviruses to express insecticidal toxins, primarily insect-specific toxins that are found in the venom of organisms that prey on insects (Maeda, S., et al., 1991, Stewart, L., et al., 1991, Windass, J., U.S. Pat. No. 5,885,569). Most of these toxins target the insect nervous system, which is also the site of activity of commercial chemical insecticides.
Although these three types of modifications have resulted in some enhancement of the speed of activity of baculovirus insecticides, chemical insecticides still act significantly faster. One limitation of all three approaches is that, just as with unmodified baculovirus insecticides, they are likely to require the establishment of a productive infection involving viral replication in order to be effective.